The present invention relates to a method for the integrated treatment of biomass from a cell culture process for producing clear cell lysate containing plasmid DNA, and a plasmid DNA cell lysate produced by the method.
Treatment is to be understood as meaning a conditioning method for providing clear cell lysate containing plasmid DNA. An integrated method is to be understood as meaning a method in which the individual steps of the method are contiguous with one another so that the product stream is conveyed virtually continuously. The basically multistage method can be carried out continuously or batchwise.
The separation of biological material from a cell culture process, the digestion of the biological material and the production of a clear cell lysate containing plasmid DNA is a customary method in the area of molecular biology. In the known methods, biological material, for example comprising E. coli bacteria cells, is separated from the culture supernatant, resuspended and then digested. Separating off the solid constituents gives a clear cell lysate which contains the plasmid DNA in addition to genomic DNA, RNA, proteins and endotoxins.
It is known that biological materials can be separated from the cell culture process by batchwise or continuous centrifuging. The separation of baker's yeast cells by filtration over a bed of filtering agent is described in GB-A-1082862. Said patent also discloses the separation of cell residues from a yeast autolysate by means of a filtering agent.
The customary methods for cell digestion are the known alkaline lysis and thermal lysis. In order to improve the cell digestion, enzymes, for example lysozyme, and/or detergents are frequently added to the cell suspension. In the alkaline lysis method, a precipitate which substantially contains the cell debris and parts of the genomic DNA and of the protein is obtained after the cell digestion at pH 12 by addition of sodium hydroxide solution and sodium dodecylsulphate and subsequent neutralization with a high molecular weight acetate buffer. The complete isolation of this precipitate can be achieved only by thorough centrifuging. A centrifugal acceleration of 12000 g is not sufficient for this purpose (I. Feliciello et al., Anal. Biochem. 212 (1993) 394-401). The precipitate can be very substantially isolated only at a centrifugal acceleration of 26000 g for 30 minutes at 4° C. On the other hand, the filtration of this precipitate is associated with low process rates and large losses of plasmid DNA, even with the aid of very fine filtering agents or in combination with a flotation step. The adsorption of plasmid DNA on the surface of the filtering agent contributes considerably to the losses. In particular, plasmid DNA binds very rapidly and strongly to such mineral surfaces if the concentration of divalent cations exceeds 0.1 mmol or the monovalent cations exceed 20 mmol in the case of potassium or 50 mmol in the case of sodium (G. Romanowski et al., Appl. Environ. Microbiol. 57 (1991) 1057-1061).
High molecular weight nucleic acids are sensitive to hear forces. Strong shear forces can lead to irreversible damage to nucleic acids, in particular to breaks in the strand. For this reason, mechanical digestion methods are seldom used for nucleic acid treatment (A. Carlson et al., Biotechnol. Bioeng. 48 (1995) 303-315). It is also known that, in industrial centrifuges with continuous introduction of liquid, high shear forces at the rotor inlet act on the nucleic acids and inevitably lead to breaks in the strand.
WO 96/36706 describes a method in which microorganisms are digested in the presence of detergents (Triton®) and by heating to 70° C. to 1000° C. in a flow-through heat exchanger. This is a combination method comprising thermolysis and with a detergent (optionally with lysozyme). In the absence of lysozyme, a thermal treatment for 30 seconds is described; in the presence of lysozyme, a thermal treatment for 6 seconds is described. On cell digestion by heating, solid compounds comprising debris, genomic DNA and proteins form. In addition, very substantial denaturing of DNA-degrading enzymes, so-called DNases, may be assumed as a result of the heat treatment. A clear cell lysate is obtained after batchwise centrifuging. In this method, only the cell digestion is carried out continuously, the solid constituents being separated off batchwise by centrifuging. In order to obtain the final clear cell lysate, a filtration over a membrane filter also had to be carried out after the centrifuging.
According to WO 92/207863 an apparatus and a method for isolation of nucleic acids from cell suspensions are known. According to this, the cells are immobilized in the cavities of an upstream, porous matrix in the form of a layer. This is achieved by deep filtration in the matrix, by virtue of the fact that the cavity size is of the order of magnitude of the cells and the matrix surface has ion exchange properties. The particle size of the matrix is 10 to 50 μm. Owing to the ion exchange properties, DNA is adsorbed on the matrix surface. The invention does not relate to adsorption of DNA. The known method gives purified DNA but not a clear cell lysate containing plasmid DNA.
EP-A-0814156 describes a method for purifying DNA. The kieselguhrs used therein are not specified. The lysis is affected by the alkaline method. Thermal lysis is not described.
The prior art thus discloses neither methods which make it possible to work up large amounts of biomass in an integrated manner nor methods by means of which large amounts of clear cell lysates containing plasmid DNA can be provided by an integrated procedure.
There is a need for an efficient conditioning method for providing clear cell lysate containing plasmid DNA from a cell culture process which permits plasmid DNA purification.